The vascular endothelium constitutes a contiguous barrier to circulating immunocompetent cells and is the initial site of allogeneic contact in a vascularized allograft. As such, its potential role as both stimulator and target of alloimmune responses leading to transplant rejection has been postulated. The overall goal of this project is to determine the molecular basis of allospecific interactions between lymphocytes and microvascular endothelial cells (EC). In vitro, lymphocyte-EC adherence can be quantitatively evaluated in a 90 minute binding assay, and subsequent EC activation assessed with cytofluorographic EC analysis for the surface expression of HLA-DR after 72 hour lymphocyte-EC coculture. Prior studies indicate that lymphocyte binding to microvascular EC is mediated via a receptor-ligand interaction. Of all mononuclear cell subsets tested, CD16+ (natural killer) lymphocytes most efficiently bind to and activate EC. Spcific proposals now include to 1) assess via extensive family and twin studies whether the degree to which allogeneic lymphocytes bind and activate human EC is a genetically determined consequence of the allogeneic combination; 2) characterize supernatants from cocultures of EC and allogeneic lymphocyte subsets with respect to their ability to activate fresh EC; 3) generate monoclonal antibodies which inhibit binding and use these antibodies to biochemically define the lymphocyte receptor(s) for EC binding and its EC ligand(s); 4) also screen antibodies for their ability to inhibit EC activation, dissecting whether activating signals transduced after binding occurs depend upon a separate set of membrane structures; 5) analyze early post-cardiac transplant biopsies immunohistochemically for the presence of CD16+ cells; 6) attempt to generate allospecific natural killer clones stimulated by genetically distinct EC lines; and 7) utilize such clones to produce anti-clonotypic monoclonal antibodies, initiating definition of clonotypic structures on these lymphoid cells which do not bear conventional T cell antigen receptors. Knowledge of surface molecules involved in lymphocyte-EC interactions should eventually allow much more specifically targeted therapy to prevent allograft rejection, thus reducing the major morbidity associated with the current immuosuppressive regimens.